The concentration of high density lipoproteins (HDLs) is inversely associated with the risk of developing premature coronary heart disease, but our understanding of the metabolic pathways that control plasma HDL concentrations is limited. HDLs exist in plasma as discrete size subfractions that can be separated by apolipoprotein content and size into small, medium, and large particles containing two, three, and four molecules of apolipoprotein A-I (apoA-l) per particle. The overall goal of this project is to elucidate the molecular pathways of HDL subfraction formation to fill the gaps in knowledge of factors that control plasma concentrations of HDL and HDL subfraction heterogeneity. Our previous studies and preliminary data have described a novel pathway for HDL subfraction metabolism in non-human primates and in human apoA-I transgenic mice in which small HDL are converted in a unidirectional pathway to medium or large HDL before being removed from plasma and catabolized by the liver. Furthermore, our data show no evidence for the production of pre-beta apoA-I during the catabolism of large HDL. In the next grant cycle, we propose to investigate the molecular pathways of HDL subfraction metabolism using transgenic and gene targeted mice to elucidate the molecular details of HDL subfraction particle assembly, intravascular metabolism, and tissue catabolism. In Specific aim 1, we will test the hypothesis that the liver and intestine are the major sites of nascent HDL assembly using hepatic and intestinal specific AbcA1 transporter knockout mice. In Specific aim 2, we will test the hypothesis that hepatic AbcA1 function is rate limiting in the assembly of lipid free apoA-I with lipid to form small nascent discoidal HDL particles. In Specific aim 3, we will test the hypothesis that AbcA1 is involved in the addition of lipid to small spherical plasma HDL and their subsequent conversion to medium and large HDL particles outside the plasma compartment. The results of these proposed studies will increase our basic understanding of the role of AbcA1 in the formation and maturation of HDL subfractions and may lead to a better understanding of ways to increase plasma HDL concentrations or stimulate reverse cholesterol transport by dietary or pharmacological methods.